Compound

ABSTRACT

A compound including a polymer is represented by general formula (1): 
       L-Y-A  (1)
         wherein, A is a single-chain antibody moiety, which is a polypeptide including an antigen-binding site, L is a linker moiety, which is a polypeptide including a protease cleavage site, Y is a peptide moiety, which includes 0 or more amino acids connecting the linker moiety L with the single-chain antibody moiety A, and the linker moiety L binds to an N terminus of the peptide moiety Y or an N terminus of the single-chain antibody moiety A.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.12/821,711 filed Jun. 23, 2010, which claims priority to Japanese PatentApplication No. 2010-109288 filed May 11, 2010, and Japanese PatentApplication No. 2009-152611 filed Jun. 26, 2009, all of which are herebyincorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compounds and a process for producingthe compounds.

2. Description of the Related Art

In order to detect lesion markers present at lesion sites, imagingcontrast agents for in vivo diagnosis have been developed. Some of theimaging contrast agents are compounds having lesion marker bindingmolecules such as antibodies labeled with physical signal generatingmolecules. As the signal generating molecules, such as radioactivenuclides, molecules which emit magnetic resonance imaging (MRI) signals,molecules which emit supersonic wave signals and molecules which emitfluorescence signals have been known. To detect lesion sites and lesionmarkers, such an imaging contrast agent is administered to a living bodyand the signal from the signal generating molecule within the agent isdetected from outside the body.

However, when such a compound as above is administered to the blood, thecompound may bind to lesion markers present at sites other than lesionsites, for example, lesion markers escaping from the lesion sites andpresent in the blood and lesion markers present at normal sites andconsequently the amount of the compound which reaches the lesion sitesmay decrease. Also, in such a case, high contrast detection of thelesion sites is difficult because the compound bound to sites other thanthe lesion sites and detected as background signal.

To solve the problem mentioned above, Lauffer et al. reported prodrugimaging contrast agents which were made biologically active in vivo inthe presence of specific biological activity (Japanese PatentApplication Laid-Open No. 2000-507577). The prodrug imaging contrastagents disclosed in this document are those in which a target moleculehaving a signal generating molecule is bound with a molecule whichreduces the binding ability of the target molecule through anenzyme-cleavable linker. The linker is cleaved in a reaction with analkali phosphatase and, as a result, the binding ability of a lowmolecular weight compound used as a target molecule with human serumalbumin increases as compared with that prior to the cleavage. Laufferet al. also reported that the cleavage of the linker changed the signalof the MRI signal generating molecule and the detection of the lesionsites were enabled with higher contrast.

Antibodies are molecules which specifically recognize antigens.Antibodies are preferably used for the detection of lesion sites ortreatment. Japanese Patent Application Laid-Open No. 2000-507577describes the possibility of using a low molecular weight compound as aimaging contrast agent but does not disclose the molecular design andthe preparation method of the imaging contrast agent, and thus theutility of the imaging contrast agent using an antibody which is madebiologically active in vivo has not been known.

SUMMARY OF THE INVENTION

When the detecting an antigen (lesion marker) present at the lesionsites is performed using a compound containing an antibody, the antibodybinds to the antigen present at sites other than lesion sites andconsequently the amount of the antibody compound which reaches thelesion sites decreases, and signals (background) from the antibodycompound bound to the antigen at sites other than the lesion sitesoccur. As a result, the detection of the lesion sites with highsensitivity and high contrast is difficult, and a compound solving thisproblem is demanded.

The compound according to a first aspect of the present inventionincluding a polymer represented by general formula (1):

L-Y-A  (1)

wherein, A is a single-chain antibody moiety, which is a polypeptideincluding an antigen-binding site; L is a linker moiety, which is apolypeptide including a protease cleavage site; Y is a peptide moiety,which including 0 or more amino acids connecting the linker moiety Lwith the single-chain antibody moiety A; and the linker moiety L bindsto an N terminus of the peptide moiety Y or an N terminus of thesingle-chain antibody moiety A.

The compound according to a second aspect of the present inventionincluding a polymer represented by general formula (2):

X-L-Y-A  (2)

wherein, A is a single-chain antibody moiety, which is a polypeptideincluding an antigen-binding site; L is a linker moiety, which is apolypeptide including a protease cleavage site; Y is a peptide moiety,which including 0 or more amino acids connecting the linker moiety Lwith the single-chain antibody moiety A; the linker moiety L binds to anN terminus of the peptide moiety Y or an N terminus of the single-chainantibody moiety A; and X is either one of polyethylene glycol, anorganic dye, an organic polymer and a particle.

The process for producing a compound according to a third aspect of thepresent invention is a process for producing a compound including apolymer represented by general formula (1), including inserting to aplasmid a nucleic acid which has a base sequence encoding a single-chainantibody moiety A, a base sequence encoding a linker moiety L and a basesequence encoding a peptide moiety Y; introducing to bacteria theplasmid in which the nucleic acids are inserted; and collecting acompound expressed by the bacteria to which the plasmid is introduced:

L-Y-A  (1)

wherein, A is single-chain antibody moiety, which is a polypeptideincluding an antigen-binding site; L is a linker moiety, which is apolypeptide comprising a protease cleavage site; Y is a peptide moiety,which including 0 or more amino acids connecting the linker moiety Lwith the single-chain antibody moiety A; and the linker moiety L bindsto an N terminus of the peptide moiety Y or an N terminus of thesingle-chain antibody moiety A.

The compound of the present invention is in a condition in which thebinding ability with the antigen in the single-chain antibody moiety isreduced at sites other than the lesion sites while the binding abilityis recovered at the lesion sites. The decrease in the amount of thesingle-chain antibody moiety which reaches the lesion sites due to thebinding with the antigens present at the sites other than the lesionsites can be suppressed by using the compound of the present invention.Also signals (background) from single-chain antibody moieties bound tothe antigens present at the sites other than the lesion sites can besuppressed by using the compound of the present invention. Therefore,high contrast detection of the lesion sites is thereby enabled by thepresent invention.

Further features of the present invention will become apparent from thefollowing description of exemplary embodiments with reference to theattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of the compound provided by the presentinvention.

FIG. 2 is a schematic view of a compound in which polyethylene glycol(PEG) is bound to the N terminus of a single-chain antibody moietythrough a linker moiety.

FIG. 3 illustrates the results of reduced type SDS-PAGE of preparedscFv-NM and scFv-WT samples with and without addition of MMP-2.

FIG. 4 illustrates the results of the measurement of interaction of theprepared scFv-NM with HER2 before and after the addition of MMP-2 usinga Biacore X system.

FIG. 5 illustrates the results of the molecular state of the preparedN-terminally PEGylated scFv-NM detected in gel filtration chromatographybefore and after the addition of MMP-2.

FIG. 6 illustrates the results of reduced type SDS-PAGE of scFv-CM whichhas been reacted with PEG-maleimide of 2 kDa, 5 kDa, 12 kDa, 20 kDa and40 kDa.

FIG. 7 illustrates the results of reduced type SDS-PAGE of the preparedC-terminally PEGylated scFv-CM with and without addition of MMP-2 and inthe presence of MMP-2 activity inhibitor.

FIG. 8 illustrates the results of reduced type SDS-PAGE of preparedscFv-WT, scFv-NM and scFv-NPM with and without addition of MMP-2.

FIG. 9 illustrates the results of the measurement of interaction withHER2 of the prepared N-terminally PEGylated scFv-NM and C-terminallyPEGylated scFv-CM PEG with and without addition of MMP-2 using a BiacoreX system.

DESCRIPTION OF THE EMBODIMENTS

Preferred embodiments of the present invention will now be described indetail in accordance with the accompanying drawings.

The First Embodiment

The compound according to the first embodiment according to the presentinvention including a polymer represented by general formula (1):

L-Y-A  (1)

wherein, A is a single-chain antibody moiety, which is a polypeptideincluding an antigen-binding site; L is a linker moiety, which is apolypeptide including a protease cleavage site; Y is a peptide moiety,which including 0 or more amino acids connecting the linker moiety Lwith the single-chain antibody moiety A; and the linker moiety L bindsto an N terminus end of the peptide moiety Y or an N terminus of thesingle-chain antibody moiety A.

The compound according to this embodiment has a single-chain antibodymoiety, a linker moiety and a peptide moiety. The linker moiety is boundto the N terminus of the peptide moiety, and the peptide moiety is boundto the N terminus of the single-chain antibody moiety. Alternatively,the linker moiety is bound to the N terminus of the single-chainantibody moiety directly (in the case where the peptide moiety has 0amino acid). In this constitution, antigen binding ability of thesingle-chain antibody moiety is inhibited or reduced, and when thelinker moiety is cleaved by protease, the antigen binding ability of thesingle-chain antibody moiety is recovered.

In this embodiment, the statement that the “antigen binding ability isinhibited or reduced” means that the ability of binding with an antigenof the single-chain antibody moiety in an unmodified state is lost orweakened by some kind of factors. In the meantime, the statement thatthe “antigen binding ability is recovered” means that the lost orweakened antigen binding ability improves to the same or similar levelas that of the single-chain antibody moiety in an unmodified state.

(Single-Chain Antibody Moiety)

In the present invention, the single-chain antibody moiety is apolypeptide comprising a site (antigen-binding site) in which a lightchain variable (VL) domain and a heavy chain variable (VH) domain of theantibody are linked with a linker comprising peptides.

Here, the antibody may be derived from human, mouse, rat, camel, birdand the other unlimited origins, and besides, it may be a chimericantibody, a polyclonal antibody or a monoclonal antibody.

The antibody mentioned above can be used without limitation, but in viewof the objects of the present invention, examples thereof includeantibodies bound to lesion markers selected from a group including EGFRfamily, VEGF family, VEGFR family, PSA, CEA, matrix metalloproteasefamily, EGF family, integrin family, selectin family, endoglin or MUCfamily and antibodies binding to HER2.

The single-chain antibody moieties can be simply and readily prepared atinexpensive cost as compared with various other antibodies and sincethey have a smaller molecular weight as compared with normal antibodies(whole antibodies, etc.), they are likely to be immediately excreted tothe outside of the body and besides they readily reach the lesion sites.Therefore, the single-chain antibody moieties are used for the detectionor treatment of the lesion sites.

The single-chain antibody moiety is, for example, a polypeptidecontaining the following amino acid sequence.

(SEQ ID NO: 1) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTV SS

Any amino acid may be linked to the C terminus of the single-chainantibody moiety.

(Linker Moiety)

In the present invention, the linker moiety refers to a polypeptidecomprising a protease cleavage site. Here, the “protease cleavage site”means a site having an amino acid sequence specifically recognized byprotease, and thereby the amino acid sequence is recognized by proteasein an activated state, and specifically hydrolyzed to be cleaved.

The protease is preferably one from matrix metalloprotease family orserine protease family, and, examples thereof include matrixmetalloprotease-2 (MMP-2), matrix metalloprotease-9 (MMP-9), prostatespecific antigen (PSA), plasmin, cathepsin, caspase as the targetprotease.

As for the protease cleavage site, polypeptides containing amino acidsequence PLGVR (SEQ ID NO: 2) can be used as a cleavage site of MMP-2.In addition, polypeptides containing amino acid sequence SSIYSQTEEQ (SEQID NO: 3) can be used as a cleavage site of PSA.

Any amino acid may be bound to the N terminus of the linker moiety.

(Peptide Moiety)

In the present invention, the peptide moiety comprises 0 or more aminoacids connecting the linker moiety with the single-chain antibodymoiety. The number of the amino acids is preferably not more than 20,and more preferably not more than 10. When the number of the amino acidsexceeds 20, when the protease cleavage site is cleaved by protease,there is a risk that the antigen binding ability of the single-chainantibody moiety may remain inhibited or reduced.

This embodiment is described in more detail by way of FIG. 1. FIG. 1 isa schematic view of the compound according to this embodiment. Thepeptide moiety is, however, not illustrated it in FIG. 1. There areshown a linker moiety 1, a single-chain antibody moiety 2, anantigen-binding site 3, an N terminus 4 and a C terminus 5 in FIG. 1.

Various kinds of mechanisms can be presumed as the mechanism to inhibitor decrease the antigen binding ability. Firstly, it is presumed thatthe linker moiety bound to the N terminus of the peptide moiety or thesingle-chain antibody moiety is present between the antigen-binding siteof the single-chain antibody moiety and antigens causing sterichindrance. In this case, it is presumed that the linker moiety covers upthe antigen-binding site of the single-chain antibody moiety, and theother antibodies cannot be linked to the antigens.

The reason for binding the linker moiety to the N terminus of thesingle-chain antibody moiety is as follows. Firstly, when the linkermoiety is bound to the linker which links a heavy chain variable (VH)domain and light chain variable (VL) domain in the single-chain antibodymoiety, the structure of the single-chain antibody moiety in itselfchanges, and the structure does not restore the original structure afterthe linker moiety is cleaved and as a result, there is a risk that theantigen binding ability might not recover. On the other hand, it ispresumed that when the linker moiety is bound to either end of thesingle-chain antibody moiety, the structure is hardly affected, andafter the linker moiety is cleaved, the single-chain antibody moietyreadily recovers the structure before the single-chain antibody moietyintroduces the linker moiety, and thus the antigen binding ability iseasy to recover after the linker moiety is cleaved. Therefore, it iseffective to introduce the linker moiety into either end of thesingle-chain antibody moiety because the antigen binding ability afterthe cleavage of the linker moiety recovers to the same or similar levelas that before the linker moiety is introduced. Next, the N terminus ofthe single-chain antibody moiety is closer to the antigen-binding sitethan the C terminus. Therefore it is presumed that when the linkermoiety is bound to the N terminus of the single-chain antibody moiety,the effects such as steric hindrance action at or around theantigen-binding site of the single-chain antibody moiety is caused moreefficiently even though the length of the linker moiety is short thanthe case where the linker moiety is bound to the C terminus of thesingle-chain antibody moiety, and that is, the antigen binding abilitycan be reduced more strongly. It is thereby presumed that inhibition orreduction of the antigen binding ability can be decreased withoutexcessively increasing the molecular weight of the compound. Suppressingthe excessive increase in the molecular weight of the compound iseffective since such suppression enables the compound to maintain thenature similar to the compound only including the single-chain antibodymoiety that the compound is readily excreted to the outside of the bodyimmediately while it is easy to reach the lesion sites. Furthermore,introducing the linker moiety to the N terminus of the single-chainantibody moiety is effective since the N terminus of the single-chainantibody moiety is close to the antigen-binding site and thus suchintroduction enables the reduction of the antigen binding ability morestrongly than the introducing to the C terminus. In addition, when thelinker moiety is introduced to the N terminus of the single-chainantibody moiety, it is possible to link other molecules to the Cterminus remote from the antigen-binding site and, for example, it ispossible to link signal generating molecules and therapeutic agentwithout impairing the antigen binding ability. It is presumed from theabove that it is the most effective to bind the linker moiety to the Nterminus of the single-chain antibody moiety.

The same holds true when the linker moiety is bound to the N terminus ofthe peptide moiety and the peptide moiety is bound to the N terminus ofthe single-chain antibody moiety.

(Signal Generating Molecule)

In the compound according to this embodiment, a signal generatingmolecule can be bound to the single-chain antibody moiety.

Various kinds of molecules can be used for the signal generatingmolecule such as radionuclides, molecules which emit MRI signals,molecules which emit supersonic wave signals and molecules which emitfluorescence signals. For example, for the signal generating moleculeswhich absorb light and give off supersonic wave signals or lightsignals, Alexa Fluor 680, Alaxa Fluor 700, Alexa Fluor 750, Alexa Fluor790 (trademarks, Invitrogen Corp.), Cy 5, Cy 5.5, Cy 7 (trademarks, GEHealth Care), HiLyte 647, HiLyte 680, HiLyte 750 (trademarks, AnaSpec,Inc), DY-680, DY-700, DY-730, DY-750, DY-782 (trademarks, Dyomics, Jena,Germany) can be used. It is also effective to select near infrared lightabsorption pigments which absorb the light of near infrared wavelengthrange of about 600 nm or more and about 1,000 nm or less excellent inliving body permeability for the molecules which absorb light and giveoff supersonic wave signals or light signals. In addition, various kindsof particles can be used for particles such as particles of metals andmetal oxides such as iron oxide particles, gold particles, goldnanorods, platinum and silver. Furthermore, it is possible to bindtherapeutic agents such as anticancer agents to the single-chainantibody moiety of this embodiment so as to deliver the therapeuticagents to the lesion sites and reduction of the side effect can beexpected by reducing the deliver of the therapeutic agents to the sitesother than the lesion sites.

Among compounds of the present invention, those to which signalgenerating molecules are linked can be also used for the detection ofthe lesion markers. That is, a process for detecting lesion markers isprovided which comprises administering to an individual a compound ofthe present invention to which a signal generating molecule is linked oradding such a compound to a sample obtained from an individual anddetecting signals, thereby detecting presence or absence of lesionmarkers or presence or absence of lesion sites generating lesion markersin the individual or in the sample obtained from an individual. Thedetection methods of the lesion markers are as follows. That is, acompound of the present invention to which a signal generating moleculeis bound is administered to an individual or added to a sample obtainedfrom an individual such as the internal organs. Here, the termindividual is not particularly limited to human or mammals such asexperimental animals or pets but refers to all the creatures and insideindividual or the sample obtained from an individual includes internalorgans, tissues and tissue slices. After the administration or addition,signals of signal generating molecules contained in the individual orthe sample are detected with a measuring instrument which can detectsignal generating molecules. As a result, when signals are detected in alevel higher the threshold values as a standard, it can be estimatedthat a lesion marker or a lesion site generating a lesion marker ispresent in the individual or that a lesion marker is present in thesample or a lesion site generating a lesion marker is present in theindividual from which the sample is derived.

(Imaging)

Similarly, among compounds of the present invention, those to which asignal generating molecule is linked can be used for imaging the lesionmarker. The imaging methods of the lesion marker are as follows. Thatis, a compound of the present invention is administered to an individualor added to a sample obtained from an individual and detecting signals,and thereby imaging as to presence/absence of a lesion marker in theindividual or the sample obtained from an individual or presence/absenceof a lesion site generating a lesion marker can be performed.

The Second Embodiment

In the following, the compound according to the second embodiment of thepresent invention is described but description about what is common withthe first embodiment is omitted.

The compound according to the second embodiment of the present inventionincluding a polymer represented by general formula (2):

X-L-Y-A  (2)

wherein, A is a single-chain antibody moiety, which is a polypeptideincluding an antigen-binding site; L is a linker moiety, which is apolypeptide comprising a protease cleavage site; Y is a peptide moiety,which including 0 or more amino acids connecting the linker moiety Lwith the single-chain antibody moiety A; the linker moiety L binds to anN terminus of the peptide moiety Y or an N terminus of the single-chainantibody moiety A; and X is either one of polyethylene glycol, anorganic dye, an organic polymer and a particle.

The compound according to this embodiment has a single-chain antibodymoiety, a linker moiety, a peptide moiety and a site (X in Formula (2))which can cover the antigen-binding site of the single-chain antibodymoiety. The linker moiety is bound to N terminus of the peptide moiety,and the peptide moiety is bound to the N terminus of the single-chainantibody moiety. Alternatively, the linker moiety is bound to the Nterminus of the single-chain antibody moiety directly (in the case wherethe peptide moiety has 0 amino acid). In this constitution, antigenbinding ability of the single-chain antibody moiety is inhibited orreduced, and when the linker moiety is cleaved, the antigen bindingability of the single-chain antibody moiety is recovered.

The site which can cover up the antigen-binding site is either one ofpolyethylene glycol (hereinbelow abbreviated as PEG), an organic dye, anorganic polymer and a particle. For the organic dye, Alexa Fluor 680,Alaxa Fluor 700, Alexa Fluor 750, Alexa Fluor 790 (trademarks,Invitrogen Corp.), Cy 5, Cy 5.5, Cy 7 (trademarks, GE Health Care),HiLyte 647, HiLyte 680, HiLyte 750 (trademarks, AnaSpec, Inc), DY-680,DY-700, DY-730, DY-750, DY-782 (trademarks, Dyomics, Jena, Germany),etc. can be used. For the organic polymer, polylactic acid (Polylacticacid, PLA), polylactic acid glycolic acid copolymer (Poly(DL-lactic-co-glycolic acid), PLGA), etc. can be used.

FIG. 2 illustrates the case where PEG is introduced as a schematic view.The peptide moiety is, however, not illustrated in FIG. 2. There areshown a linker moiety 1, a single-chain antibody moiety 2, anantigen-binding site 3, an N terminus 4, a C terminus 5 and PEG 6 inFIG. 2. It is supposed that it is effective that the site which cancover the linker moiety and the linked antigen-binding site is bound tothe N terminus of the single-chain antibody moiety even when thesemolecules are use. For example, PEG is particularly preferably used asthe site which can cover up the antigen-binding site, and it is supposedthat PEG is present in a sterically expanded state in an aqueoussolution. Therefore, it is presumed that when the linker moiety and thelinked PEG were introduced to the N terminus of the single-chainantibody moiety, PEG is present between the antigen-binding site of thesingle-chain antibody moiety and the antigens, it causes larger sterichindrance, as compared with, for example, the case where it isintroduced to the other site than the N-terminal site such as C-terminalsite and thereby the action to inhibit or reduce the antigen bindingability becomes larger, and it is enabled to reduce the antigen bindingability of the single-chain antibody moiety more strongly.

Various kinds of effects can be expected from the introduction of PEG inaddition to the inhibition of the antigen-binding ability, etc. Examplesof such effects include regulation of the molecular weight of thecompound and regulation of the protein adsorption ability of thecompound by introducing PEG. Since the linker moiety is not cleaved andPEG is not detached from the compound having PEG introduced thereto inblood before the compound reaches the lesion site, the molecular weightof the compound is maintained as large and the protein adsorbing abilityof the compound is suppressed. Thereby, the compound is maintained at asize larger than that to be excreted from the kidney and the loss of thecompound by the excretion from the kidney is suppressed. The loss of thecompound by excretion via such as liver which removes the compound tothe outside of the body through adsorption of protein is alsosuppressed. On the other hand, the linker moiety is cleaved at thelesion sites, and the molecular weight of the compound decreases becausePEG is detached. This would also enhance the penetrative properties ofthe compound at the lesion sites as compared with the sites other thanthe lesion sites so that the compound may penetrate to the depth of thesites and thereby enable improvement in the accumulation of the compoundat the lesion sites. In contrast, the compound does not penetrate to thedepth of the normal sites since the linker moiety is not cleaved and PEGis not detached at the normal sites. On this account, it would bepossible that signals (background) which occur by the accumulation ofthe compound at the normal sites is reduced.

It is preferable that the molecular weight of PEG is not less than 20kDa. It is also preferable that the molecular weight of PEG is not morethan 40 kDa.

In the case where the enzyme which cleaves the linker moiety isidentical with the lesion marker to which the single-chain antibodymoiety binds, it would be possible that both the presence and theactivity of the enzyme are detected at the same time. In addition, whenthe enzyme which cleaves the linker moiety is specific to the lesionsites and the enzyme is different from the lesion marker to which thesingle-chain antibody moiety binds, the compound only accumulates at thelesion sites only in the case where the two kinds of the lesion markersat the lesion sites are present, it would be possible that the positionprecision is improved and a detailed position information of the lesionsites is detected. For example, in the case of combination of asingle-chain antibody moiety which binds to HER2 and a linker moietywhich contains a cleavage site of MMP-2, it would be possible thattumors which show both the two conditions that HER2, poor prognosisfactor of cancers, is positive and that MMP-activity as an index showingproperties such as metastasis and invasion is high, that is, tumorsappearing to be highly malignant, are detected with high positionaccuracy. In this case, soluble HER2 present in blood or the other fluidand HER2 positive/MMP-2 negative tumor sites are hard to be detectedwith the imaging contrast agent, it would be enabled that background isreduced. In addition, in the case of combination of a single-chainantibody moiety which binds to vascular endothelium growth factor VEGFand a linker moiety which contains a cleavage site of MMP-2, since thepresent compound is hard to bind to the VEGF seeping into blood while itis easy to bind to the VEGF present in high concentration at lesionsites where activated MMP-2 is present, lesion sites where angiogenesisis active and VEGF is present in high concentration are detected withhigh position accuracy.

The Third Embodiment

The third embodiment of the present invention is described. Thisembodiment relates to a process for producing the compound comprising apolymer represented by the general formula (1) mentioned above.Specifically, the process has the following steps.

(i) A step of inserting to a plasmid a nucleic acid which has a basesequence encoding a single-chain antibody moiety A, a base sequenceencoding a linker moiety L and a base sequence encoding a peptide moietyY;(ii) A step of introducing to bacteria the plasmid in which the nucleicacid is inserted; and(iii) A step of collecting a compound expressed by the bacteria to whichthe plasmid is introduced.

Here, the process for producing the compound according to thisembodiment may include a step other than the steps (i) to (iii)mentioned above.

EXAMPLES

In the following, the present invention is described by way of examplesso as to clarify the characteristic of the present invention in moredetail. It should be noted, however, that the other combinations of themoieties of the single-chain antibody moiety, the linker moiety and thesite which can cover the antigen-binding site can be used in the presentinvention and that the present invention is not limited to theseexamples.

Example 1 Preparation of hu4D5-8 scFv

Firstly, based on the gene sequence of the variable region of IgG whichbinds to HER2, the gene fragment which encoded a single-chain antibody(scFv) moiety was prepared. An amino acid sequence PLGVR which wasspecifically cleaved by MMP-2 was bound to the N terminus of theprepared gene, and a 6×His tag comprising 6 consecutive histidines forprotein purification was bound to the C terminus and further 2 glycinesas a spacer and a cysteine for introducing a signal generating moleculewere disposed in the downstream thereof. As a comparison, a gene whichdid not contain in the C terminus the amino acid sequence PLGVR which isspecifically cleaved by MMP-2 was prepared in the same way. A plasmidpET-22b (+) (Novagen Corporation) in which the above gene fragment wasinserted in the downstream of the T7 promoter was introduced into E.coli (Escherichia coli BL21 (DE3) to obtain a strain for expression.After the obtained strain was cultivated in 4 ml of LB-Amp culturemedium overnight, the whole volume was added to 250 ml of 2×YT culturemedium and subjected to shaken cultivation at 120 rpm, 28° C. for 8hours. Then, IPTG was added in the final concentration of 1 mM and themixture was cultivated at 28° C. overnight. The cultivated E. coli wascentrifuged at 8000×g, 4° C. for 30 minutes and the supernatant of theculture solution was collected. Ammonium sulfate was added to theobtained culture solution in an amount of 60% by weight and the proteinswere precipitated by salting out. After the salted out solution was leftstandstill at 4° C. overnight, sediments were collected by centrifugingthe solution at 8000×g, 4° C. for 30 min. The obtained sediments weredissolved in 20 mM Tris.HCl/500 mM NaCl buffer, and 1 L of the bufferwas dialyzed. The protein solution after the dialysis was added to acolumn filled with His.Bind (registered trademark) and Resin (NovagenCorporation) and purified by metal chelate affinity chromatography withNi ion. It was confirmed that the purified hu4D5-8 scFv with an MMP-2substrate introduced at the N terminus (scFv-NM) and hu4D5-8 scFvwithout an MMP-2 substrate at the N terminus (scFv-WT) showed a singleband and had a molecular weight of about 28 kDa in SDS-PAGE.

The prepared scFv-NM had the following sequence.

(SEQ ID NO. 4) MSGPLGVRGAMDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAAALEHHHHHHGGC

The prepared scFv-WT had the following sequence.

(SEQ ID NO. 5) MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT VSSAAALEHHHHHHGGC

Example 2 Confirmation of Cleavage of scFv-NM at the Linker Moiety bythe Addition of MMP-2

Change in the molecular weight resulted from the cleavage of the peptideby the addition of MMP-2 to the scFv-NM prepared above was measured.0.10 mg/ml of an active type MMP-2 (Cosmo Bio Co., Ltd.) was added inabout 15 nM to about 4 μM of PEGylated scFv dialyzed in a TCNB buffer(50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij 35, pH 7.5), and thereaction was performed for about 20 hours and the change in themolecular weight was measured by reduced type SDS-PAGE. As a result,decrease in the molecular weight was detected in scFv-NM by adding MMP-2as shown in FIG. 3. On the other hand, decrease in the molecular weightwas not detected in scFv-WT. It was confirmed from the above that thepeptide of scFv-NM was cleaved by MMP-2.

Example 3 Confirmation of Improvement in the Antigen Binding Ability ofscFv-NM by the Addition of MMP-2

The interaction of scFv-NM, scFv-WT prepared above with HER2, which wasan antigen, was measured using Biacore X system (GE Healthcare Co.,Ltd.) and the change in the binding ability of scFv before and after theaddition of MMP-2 was measured. As an antigen, Recombinant HumanErbB2/Fc Chimera (with R & D Systems, Inc.) was immobilized to thecarboxymethyldextran chain on the surface of CM-5 chip by amine couplingaccording to the recommendation of the manufacture. The immobilizedamount was about 1,000 R U. As a running buffer, PBS-T (2.68 mM KCl/137mM NaCl/1.47 mM KH₂PO₄/1 mM Na₂HPO₄/0.005% Tween 20, pH 7.4) was usedand 50 nM scFv was injected under a condition of 20 μL/min in the flowrate, and the binding ability was evaluated. As a result, as shown inFIG. 4, change in the sensorgram was able to be confirmed before andafter the addition of MMP-2 and it was found that the antigen bindingability of scFv-NM was improved. On the other hand, it was confirmedthat the antigen binding ability of scFv-WT hardly changed in MMP-2before and after the addition of MMP-2.

Example 4 Preparation of scFv-NM with PEGylated N Terminus

After the buffer of the scFv-NM prepared in Example 1 was substitutedwith 100 mM phosphate buffer (pH 5.0), PEG-aldehyde (Nichiyu Co., Ltd.)of 5 kDa, 20 kDa or 40 kDa in 40-time molar amount was added to thescFv-NM. 2-Picoline borane was added in the final concentration of 20 mMand the reaction was performed at 4° C. for about 3 days. After thereaction, the buffer was changed to a TCNB buffer (50 mM Tris, 10 mMCaCl₂, 150 mM NaCl, 0.05% Brij 35, pH 7.5) and the fraction in which 1molecular of PEG was supposed to be bound was separated by gelfiltration chromatography with Superdex 200 GL 10/300 column (GEHealthcare Co., Ltd.) and scFv-NM in which the N terminus was supposedto be PEGylated was obtained.

Example 5 Confirmation of the Detachment of PEG by the Addition of MMP-2from scFv-NM with PEGylated N Terminus

Change in the molecular weight of the scFv-NM prepared above in whichthe N terminus was supposed to be PEGylated was evaluated by theaddition of MMP-2. 0.10 mg/ml of an active type MMP-2 (Cosmo Bio Co.,Ltd.) was added in about 10 nM in the final concentration to the scFv-NMin which the N terminus was supposed to be PEGylated, which wasdissolved in a TCNB buffer and the reaction was performed for about 20hours and the change in the molecular weight was evaluated by gelfiltration chromatography with Superdex 200 GL 10/300 column. The resultof the gel filtration chromatography (absorption at 280 nm monitored) of20-kDa PEGylated scFv-NM before and after the addition of MMP-2 wasshown in FIG. 5. Two peaks which were not present before the cleavagewere detected by the addition of MMP-2 as shown in FIG. 5. These areconsidered as a monomer and a dimer of scFv-NM not modified with PEG.That is, this result shows that PEG, which had modified the N terminus,was detached from scFv-NM by the cleavage by MMP-2 of MMP-2 substrateswhich was introduced into the N terminus. That is, it was able to beconfirmed that the N terminus was modified with PEG. In addition, as aresult of comparing the ratio of the peak area which was supposed tocorrespond to the scFv-NM from which PEG was detached and the total peakarea, the ratio that the N terminus was PEGylated among the wholePEGylated scFv-NM PEG was around 50%. As for the remaining 50%, theamino group of the lysine residue in scFv-NM molecules was supposed tobe PEGylated. It was also confirmed that about 50% of the N terminuswere similarly PEGylated when PEG aldehyde of 5 kDa or 40 kDa was used.It was confirmed from above that scFv-NM with PEGylated N terminus wasacquired.

Example 6 Preparation of scFv in which a MMP-2 Substrate was Introducedinto the C Terminus

Based on gene sequencing of the variable region of IgG which binds toHER2, the gene fragment which encoded a single-chain antibody (scFv)moiety was prepared. An amino acid sequence PLGVR which was specificallycleaved by MMP-2, a cysteine residue for linking a molecule whichreduced the antigen-binding ability and a 6×His tag comprising 6consecutive histidines for protein purification were disposed in the Cterminus of the prepared gene. A plasmid pET-22b (+) (NovagenCorporation) in which the above gene fragment was inserted in thedownstream of the T7 promoter was introduced into E. coli (Escherichiacoli BL21 (DE3)) to obtain a strain for expression. After the obtainedstrain was cultivated in 4 ml of LB-Amp culture medium overnight, thewhole volume was added to 250 ml of 2×YT culture medium and subjected toshaken cultivation at 120 rpm, 28° C. for 8 hours. Then, IPTG was addedin the final concentration of 1 mM and the mixture was cultivated at 28°C. overnight. The cultivated E. coli was centrifuged at 8000×g, 4° C.for 30 minutes and the supernatant of the culture solution wascollected. Ammonium sulfate was added to the obtained culture solutionin an amount of 60% by weight and the proteins were precipitated bysalting out. After the salted out solution was left standstill at 4° C.overnight, sediments were collected by centrifuging the solution at8000×g, 4° C. for 30 min. The obtained sediments were dissolved in 20 mMTris.HCl/500 mM NaCl buffer, and 1 L of the buffer was dialyzed. Theprotein solution after the dialysis was added to a column filled withHis.Bind (registered trademark) and Resin (Novagen Corporation) andpurified by metal chelate affinity chromatography with Ni ion. It wasconfirmed that the purified scFv with MMP-2 substrate introduced at theC terminus (scFv-CM) showed a single band and had a molecular weight ofabout 28 kDa in SDS-PAGE.

The prepared scFv-CM had the following sequence.

(SEQ ID NO.6) MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT VSSAAAGPLGVRGCLEHHHHHH

Example 7 Preparation of scFv-CM with PEGylated C Terminus

After the buffer of the scFv-CM prepared above was substituted with aphosphate buffer containing 5 mM EDTA (2.68 mM KCl/137 mM NaCl/1.47 mMKH₂PO₄/1 mM Na₂HPO₄/5 mM EDTA, pH 7.4), the resultant solution wassubjected to reduction treatment with 10- to 20-time molar amount oftris(2-carboxyethyl)phosphine hydrochloride (TCEP) at 25° C. for 2 to 4hours. This scFv-CM subjected to reduction treatment was reacted withPEG maleimide (Nichiyu Co., Ltd.) of 2 kDa, 5 kDa, 12 kDa, 20 kDa or 40kDa in 10-time molar amount at 25° C. for 2 to 4 hours. It was confirmedby reduced type SDS-PAGE that these were PEGylated (FIG. 6). After thereaction, unreacted PEG-maleimide was removed by gel filtrationchromatography with Superdex 200 GL 10/300 column to obtain scFv-CM withPEGylated C terminus.

Example 8 Confirmation of the Detachment of PEG by the Addition of MMP-2from scFv-CM with PEGylated C Terminus

For those appended with 20 kDa PEG-maleimide among the scFv-CM withPEGylated C terminus prepared above, change in the molecular weight wasevaluated by the addition of MMP-2. An active type MMP-2 (0.10 mg/ml)was added in about 10 nM in the final concentration to the scFv-CM withPEGylated C terminus dialyzed in a TCNB buffer and the reaction wasperformed for 2 hours and 20 hours and the change in the molecularweight at the respective time point was evaluated by reduced typeSDS-PAGE. Another experiment in which 1 mM of 1,10-phenanthroline(Sigma-aldrich), an MMP-2 activity inhibitor was added and the reactionwas performed for 20 hours in the same way. As a result, a band wasdetected in the vicinity of 28 kDa which was the molecular weight ofscFv-CM in 2 hours after the addition of MMP-2, and almost scFv-CM wasdetected as a main band in 20 hours as shown in FIG. 7. The detection ofthis scFv-CM band was suppressed by the addition of 1,10-phenanthroline.It was confirmed from above that PEG of scFv-CM with PEGylated Cterminus was specifically cleaved and detached from scFv-CM by MMP-2.

Example 9 Preparation of scFv in which a Peptide and an MMP Substratewas Introduced to the N Terminus

Firstly, based on the gene sequence of the variable region of IgG whichbinds to HER2, the gene fragment which encoded a single-chain antibody(scFv) moiety was prepared. A peptide GGGSGGGS (SEQ ID NO:8), and in thedownstream thereof an amino acid sequence PLGVR which was specificallycleaved by MMP-2 were disposed in the N terminus of the prepared gene,and in the downstream thereof a 6×His tag comprising 6 consecutivehistidines for protein purification and further 2 glycines as a spacerand a cysteine for introducing a signal generating molecule weredisposed in the C terminus. A plasmid pET-22b (+) (Novagen Corporation)in which the above gene fragment was inserted in the downstream of theT7 promoter was introduced into E. coli (Escherichia coli BL21 (DE3)) toobtain a strain for expression. After the obtained strain was cultivatedin 4 ml of LB-Amp culture medium overnight, the whole volume was addedto 250 ml of 2×YT culture medium and subjected to shaken cultivation at120 rpm, 28° C. for 8 hours. Then, IPTG was added in the finalconcentration of 1 mM and the mixture was cultivated at 28° C.overnight. The cultivated E. coli was centrifuged at 8000×g, 4° C. for30 minutes and the supernatant of the culture solution was collected.Ammonium sulfate was added to the obtained culture solution in an amountof 60% by weight and the proteins were precipitated by salting out.After the salted out solution was left standstill at 4° C. overnight,sediments were collected by centrifuging the solution at 8000×g, 4° C.for 30 min. The obtained sediments were dissolved in 20 mM Tris-HCl/500mM NaCl buffer, and 1 L of the buffer was dialyzed. The protein solutionafter the dialysis was added to a column filled with His.Bind(registered trademark) and Resin (Novagen Corporation) and purified bymetal chelate affinity chromatography with Ni ion. It was confirmed thatthe purified hu4D5-8 scFv with an purified N terminus peptide and anMMP-2 substrate introduced at the N terminus (scFv-NPM) showed a singleband and had a molecular weight of about 28 kDa in SDS-PAGE.

The prepared scFv-NPM had the following sequence.

(SEQ ID NO. 7) MSGGGSGGGSGPLGVRGAMDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAAALEHHHHHHGGC

Example 10 Confirmation of Cleavage of scFv-NPM at the Linker Moiety bythe Addition of MMP-2

Change in the molecular weight resulted from the cleavage of the peptideby the addition of MMP-2 to the scFv-NPM prepared above was measured.0.10 mg/ml of an active type MMP-2 was added in about 10 nM to about 4μM of PEGylated scFv dialyzed in a TCNB buffer, and the reaction wasperformed for about 20 hours and the change in the molecular weight wasmeasured by reduced type SDS-PAGE. As a result, decrease in themolecular weight was detected in scFv-NPM by adding MMP-2 as shown inFIG. 8. scFv-NM and scFv-CM are also shown for comparison. On the otherhand, decrease in the molecular weight was not detected in scFv-WT. Itwas confirmed from the above that the peptide of scFv-NPM was cleaved byMMP-2.

Example 11 Confirmation of the Change in the Binding Ability of scFv bythe Addition of MMP-2

The interaction of various scFv prepared above with HER2, which was anantigen, was measured using Biacore X system (GE Healthcare Co., Ltd.)and the change in the binding ability of various scFv before and after20 hours of the addition of MMP-2 was measured. As an antigen,Recombinant Human ErbB2/Fc Chimera (with R & D Systems, Inc.) was usedfor immobilization to the carboxymethyldextran chain on the surface ofCM-5 chip by amine coupling according to the recommendation of themanufacture. The immobilized amount was about 1000 to 1300 RU. As arunning buffer, PBS-T (2.68 mM KCl/137 mM NaCl/1.47 mM KH₂PO₄/1 mMNa₂HPO₄/0.005% Tween 20, pH 7.4) was used and samples prepared in 5 to100 nM were injected under a condition of 20 μL/min in the flow rate,and the binding ability was evaluated. The binding ability wereevaluated for scFv-CM without the addition of MMP-2, samples in whichPEG-maleimide of 2, 5, 12, 20 or 40 kDa was introduced to the C terminusof scFv-CM without the addition of MMP-2, a samples in whichPEG-maleimide of 40 kDa was introduced to the C terminus of scFv-CMafter the addition of MMP-2, scFv-NM with or without the addition ofMMP-2, scFv-NPM with or without the addition of MMP-2, samples in whichPEG-aldehyde of 5, 20 or 40 kDa was introduced to the N terminus ofscFv-NM with or without the addition of MMP-2, as the samples whosebinding ability was evaluated. In FIG. 9, the results of interactionwith HER2 measured for N-terminally PEGylated scFv-NM, C-terminallyPEGylated scFv-CM prepared with or without the addition of MMP-2 usingthe Biacore X system are shown by logarithm of the obtained associationrate constant (kon) in the vertical axis and molecular weight of PEG inthe horizontal axis. As shown in FIG. 9, It was confirmed thatintroduction of PEG to the N terminus was able to reduce kon moresignificantly than the introduction to the C terminus and that kon wasable to be changed by the addition of MMP-2. Specifically, scFv-NM inwhich 5 kDa PEG was introduced to the N terminus showed a value of koncomparable to scFv-CM in which 12 kDa PEG was introduced to the Cterminus, and thus it was confirmed that kon was able to be decreasedmore significantly with a low molecular weight PEG in the N terminusthan in the C terminus. In addition, the degree of change in kon by theaddition of MMP-2 is about 5 times in scFv-CM in which 40 kDa PEG wasintroduced to the C terminus while the value is about 15 times inscFv-NM in which 20 kDa PEG was introduced to the N terminus, and thusit was confirmed that the binding ability was able to be changed moresignificantly in the N terminus than in the C terminus. Furthermore, itwas confirmed that the change in kon was comparable when the change inkon for scFv-NM and scFv-NPM before and after the addition of MMP-2 wascompared.

Example 12 Preparation of Conjugates of scFv-NM with PEGylated NTerminus and Iron Oxide Particles

The scFv-NM with PEGylated N terminus having 5 kDa, 20 kDa or 40 kDa PEGwhich were prepared in example 4 were reduced in 100 mM phosphate buffer(pH 7.4) at 25° C. for 2 hours under existence of TCEP in 10-timie molaramount. The reduced scFv-NM with PEGylated N terminus was reacted withiron oxide particles containing PEG300 (nanomag-D-SPIO manufactured byCorefront Corporation) of which surface is modified with maleimide groupin 1/2000-timie molar amount at 4° C. overnight. After the reaction,scFv-NM with PEGylated N terminus which was not bound to the particleswas removed by ultrafiltration using Amicon Ultra-4 (MilliporeCorporation) of which pore size was 100 kDa. L-cysteine was added to thefiltrated product at 1 mM as the final concentration to blocknon-reacted maleimide group on the particles. Conjugates of scFv-NM withPEGylated N terminus and iron oxide particles were obtained by removingnon-reacted L-cysteine by gel filtration using PD-10 column (GEHealthcare).

Average hydrodynamic diameters of the obtained conjugates of scFv-NMwith PEGylated N terminus and iron oxide particles having 5 kDa, 20 kDaor 40 kDa PEG were determined to be 185, 188 and 192 nm respectively bydynamic light scattering techniques. On the other hand, averagehydrodynamic diameter of iron oxide particles reacted with L-cysteinewas about 174 nm, which was smaller than the conjugates. Therefore, itwas confirmed that scFv-NM with PEGylated N terminus was immobilized onthe iron oxide particle and that conjugates of scFv-NM with PEGylated Nterminus and iron oxide particles were obtained.

While the present invention has been described with reference toexemplary embodiments, it is to be understood that the invention is notlimited to the disclosed exemplary embodiments. The scope of thefollowing claims is to be accorded the broadest interpretation so as toencompass all such modifications and equivalent structures andfunctions.

This application claims the benefit of Japanese Patent Application No.2009-152611, filed Jun. 26, 2009, and Japanese Patent Application No.2010-109288, filed May 11, 2010 which are hereby incorporated byreference herein in their entirety.

What is claimed is:
 1. A process for detecting a lesion marker,comprising administering to an individual a compound comprising apolymer represented by formula (1) to a single-chain antibody moiety ofwhich a signal generating molecule is linked or adding such a compoundto a sample obtained from an individual and detecting signals, therebydetecting presence or absence of lesion markers or presence or absenceof lesion sites generating lesion markers in the individual or in thesample obtained from an individual, wherein the compound comprising thepolymer represented by general formula (1) is as follows:L-Y-A  (1) wherein, A is a single-chain antibody moiety, which is apolypeptide comprising an antigen-binding site; L is a linker moiety,which is a polypeptide comprising a protease cleavage site; Y is apeptide moiety, which comprises 0 or more amino acids connecting thelinker moiety L with the single-chain antibody moiety A; and the linkermoiety L binds to an N terminus of the peptide moiety Y or an N terminusof the single-chain antibody moiety A.
 2. A process for producing acompound comprising a polymers represented by general formula (1),comprising inserting to a plasmid a nucleic acid which has a basesequence encoding a single-chain antibody moiety A, a base sequenceencoding a linker moiety L and a base sequence encoding a peptide moietyY; introducing to bacteria the plasmid in which the nucleic acid isinserted; and collecting a compound expressed by the bacteria to whichthe plasmid is introduced:L-Y-A  (1) wherein, A is single-chain antibody moiety, which is apolypeptide comprising an antigen-binding site; L is a linker moiety,which is a polypeptide comprising a protease cleavage site; Y is apeptide moiety, which comprises 0 or more amino acids connecting thelinker moiety L with the single-chain antibody moiety A; and the linkermoiety L binds to an N terminus of the peptide moiety Y or an N terminusof the single-chain antibody moiety A.